Immersion objective lens, retention mechanism for immersion medium, and manufacturing method thereof

ABSTRACT

There is provided a retention mechanism for an immersion medium, for use in a device which observes/measures a sample by use of an immersion objective lens, includes a member which retains the immersion medium near a tip portion of the objective lens, wherein the member is configured to include at least first and second materials.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation Application of U.S. Ser. No. 11/505,164, filedAug. 16, 2006, which is a Continuation Application of PCT ApplicationNo. PCT/JP2005/002255, filed Feb. 15, 2005, which was published underPCT Article 21 (2) in Japanese, the entire contents of each of which areincorporated herein by reference.

This application is based upon and claims the benefit of priority fromprior Japanese Patent Application No. 2004-038500, filed Feb. 16, 2004,the entire contents of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an immersion objective lens in a devicewhich monitors and measures a sample with use of the immersion objectivelens, a retention mechanism for holding an immersion medium, and amanufacturing method thereof.

2. Description of the Related Art

When a sample in a micro-plate set on a sample stage is observed ormeasured by use of an immersion objective lens, an immersion medium issupplied to the tip of the objective lens, and the sample is observedand measured through the immersion medium and the bottom glass of themicro-plate. Water or immersion oil is used as this immersion medium.

The specification of U.S. Pat. No. 6,071,748 discloses a techniqueconcerning a fluorescence correlation microscopy apparatus. In thisapparatus, light is irradiated on a sample through an immersionobjective lens from the lower surface of a micro-plate, and fluorescencefrom the sample is detected. Meanwhile, the micro-plate has many wellsto contain samples, provided at positions spatially apart from oneanother. Therefore, measurement using a micro-plate involves a wideobservation range and a long distance movement in the horizontaldirection. Therefore, as a measurement area shifts, an immersion mediumat the tip portion of the lens sticks to the bottom plate of themicro-plate and is scattered, in some cases. If measurement takes a longtime, drying is accelerated and the immersion medium is lost since astrong laser beam runs through the objective lens for a long time, inother cases.

Hence, the pamphlet of International Publication No. WO 02/093232discloses a device for supplying an immersion medium which supplies alarge amount of immersion medium.

BRIEF SUMMARY OF THE INVENTION

A retention mechanism for an immersion medium, for use in a device whichobserves/measures a sample by use of an immersion objective lens,according to a first aspect of the present invention includes: a memberwhich retains the immersion medium near a tip portion of the objectivelens, wherein the member is configured to include at least first andsecond materials.

An immersion objective lens according to a second aspect of the presentinvention includes: a lens ring portion which has at least a part havinga low affinity with an immersion medium.

A method of manufacturing a retention mechanism for an immersion medium,for use in a device which observes/measures a sample by use of animmersion objective lens, according to a third aspect of the presentinvention includes: manufacturing by locating a material having a highor low affinity with the immersion medium at a member which retains theimmersion medium, or by performing a surface treatment on a materialhaving a high or low affinity with the immersion medium.

Advantages of the invention will be set forth in the description whichfollows, and in part will be obvious from the description, or may belearned by practice of the invention. Advantages of the invention may berealized and obtained by means of the instrumentalities and combinationsparticularly pointed out hereinafter.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

The accompanying drawings, which are incorporated in and constitute apart of the specification, illustrate embodiments of the invention, andtogether with the general description given above and the detaileddescription of the embodiments given below, serve to explain theprinciples of the invention.

FIG. 1 is a diagram showing the structure of an observation/measurementdevice to which a retention mechanism for an immersion medium accordingto the first embodiment of the present invention is applied;

FIG. 2 is an enlarged cross-sectional view showing a tip portion of anobjective lens and a part of a micro-plate;

FIG. 3 is a perspective view showing the retention mechanism for animmersion medium, according to the first embodiment;

FIG. 4 is a longitudinal cross-sectional view of the retention mechanismfor an immersion medium, according to the first embodiment;

FIG. 5A is a view schematically showing a contact angle between asubstrate and an immersion medium;

FIG. 5B is also a view schematically showing a contact angle between asubstrate and an immersion medium;

FIG. 6 is a cross-sectional view of a liquid pool plate;

FIG. 7 is an enlarged cross-sectional view showing a tip portion of anobjective lens and a part of a micro-plate;

FIG. 8 is a perspective view showing a retention mechanism for animmersion medium, according to a modification of the first embodiment;

FIG. 9 is a cross-sectional view showing the retention mechanism for animmersion medium, according to the modification of the first embodiment;

FIG. 10 is a perspective view showing a retention mechanism for animmersion medium, according to the fourth embodiment; and

FIG. 11 is a perspective view showing another retention mechanism for animmersion medium, according to the fourth embodiment.

DETAILED DESCRIPTION OF THE INVENTION

[First Embodiment]

FIG. 1 is a view showing the structure of an observation/measurementapparatus to which an immersion medium retention mechanism according tothe first embodiment of the present invention is applied. Thefundamental apparatus structure of this observation/measurementapparatus is based on an inverted microscope. For example, the structureand operation of the apparatus applied to, for example, a confocaloptical microscope will now be described with reference to FIG. 1.

The present observation/measurement apparatus is provided with two kindsof light sources 1 a and 1 b. For the light source 1 a, a helium neonlaser (having an output power of 2 mW and a wavelength of 633 nm) isused. For the light source 1 b, an argon-ion laser (having an outputpower of 10 mW and a wavelength of 488 nm) is used.

The traveling direction of a light beam emitted from the light source 1a is changed by a mirror 2, and is further synthesized with another beamemitted from the light source 1 b by a dichroic mirror 3, to be unifiedinto one light path. The one light beam thus synthesized becomes acollimated beam whose beam diameter is enhanced by a lens 4. Thecollimated beam is reflected by another dichroic mirror 5 and reaches anobjective lens 10.

The dichroic mirror 5 is manufactured in a manner that one side of aglass-made flat plate is subjected to multi-layer film coating, tooptimize spectral characteristics of transparency and reflection. A cubebeam splitter is used as the dichroic mirror 5.

On an XY stage 19, a micro-plate 20 holding samples is fixed and set.The position of the micro-plate 20 in a horizontal plane is adjusted bythe XY stage 19, and the position of the objective lens 10 in thevertical direction is adjusted by a Z-axis adjustment mechanism 17.Thus, the samples as targets to measure are adjusted to be positioned inan area where the objective lens 10 converges light (the area is calleda confocal area).

The micro-plate 20 has 96 wells 22 (circular grooves to containsamples). The bottom face of each well 22 is formed, as a window, of amaterial such as glass which allows visible light to pass. Light emittedfrom the objective lens 10 opposed thereto below the micro-plate 20penetrates this window and converges inside a sample.

Used as the objective lens 10 is, for example, a X40 water immersionobjective lens (NA 0.9). Therefore, water as an immersion medium isfilled between the bottom face of the micro-plate 20 (e.g., the bottomface of the wells 22) and the tip portion of the objective lens 10. Thesize and shape of the confocal area which the light emitted form lightsources forms in a well 22 has a substantially cylindrical shape havinga diameter of about 0.6 μm and a length of 2 μm. The convergenceposition of the laser beam is substantially the center part of the well22 in the horizontal direction (X-Y axis), and is 100 μm above theposition of the upper wall of the bottom face of the well 22.

The laser beam converged by the objective lens 10 excites a fluorescentmaterial in samples, and the fluorescent substance emits fluorescence.Various fluorescent pigments may be used as the fluorescent substance.For example, when rhodamine green (RhG) is used, a peak absorptionwavelength of rhodamine green is near 490 nm, and a peak light emissionwavelength thereof is near 530 nm. Hence, rhodamine green is excited byan argon laser. Alternatively, when Cy5 is used as the fluorescentsubstance, this substance is excited by a helium neon laser. Cy5 has apeak absorption wavelength near 640 nm and a peak light emissionwavelength near 670 nm.

The fluorescence passes through the objective lens 10 again andsubsequently through the dichroic mirror 5, and enters into a barrierfilter 7. The barrier filter 7 has a disk-like shape. The transparencycharacteristic of the barrier filter 7 is adjusted so as to allow onlythe light within a wavelength range of a fluorescent light emissionspectrum to pass. As a result, a part of the laser beam scatters withina sample container or is reflected by the wall of the well 22, so thatnoise light returning to an incident path can be cut. That is, thewavelength of fluorescence and the wavelength of background light aredifferent from each other.

Signal light which has passed through the barrier filter 7 passesthrough a lens 8, forming converged light, which is reflected by amirror 9. The light is then converged on a pinhole plane of a pinhole 16provided in the rear side. That is, the focal plane of the lens 8 andthe open face of the pinhole 16 agree with each other. The pinhole 16has a diameter of 50 μm. Background light from the other area than theconfocal area of light which is formed in the well 22 is removed by thepinhole 16.

A light detector 11 is located near and in the rear side of the pinhole16. Signal light received by the light detector 11 is low light leveland forms a photon pulse. Hence, a faint light detector such as anavalanche photodiode (APD) or an photoelectron multiplier tube is usedas the light detector 11.

The light detector 11 converts the signal light into an electric signal(current pulse) and inputs the signal to a signal processing device 12.In the signal processing device 12, the electric signal is amplified andsubjected to waveform shaping, thereby to form an on-off voltage pulsewhich is guided to a computer 13. The on-off voltage pulse is storedinto a memory (not shown) of the computer 13. Based on this data,calculation such as correlation analysis is carried out. Further,fluorescent intensity, fluorescent lifetime, an autocorrelation functionof intensity fluctuation of obtained fluorescence, or analysis resultsuch as a cross-correlation function are displayed on a monitor 14 ofthe computer 13.

FIG. 2 is an enlarged view of the part A shown in FIG. 1, e.g., anenlarged cross-sectional view showing the tip portion of the objectivelens 10 and a part of the micro-plate 20.

As described in FIG. 1, the micro-plate 20 is set on the XY stage 19(not shown). The objective lens 10 is provided up side down, facing thebottom face of the micro-plate 20. Samples 23 are contained in wells 22recessed in the micro-plate 20. In size the micro-plate 20 is 127 mmwide, 85.5 mm long, and 14.5 mm high. In the case of 96 circular wellswhich are frequently used, the groove has a diameter of 6.6 mm and adepth of 12.5 mm.

An immersion medium 31 (for example, immersion water) is filled betweenthe tip portion of the objective lens 10 and the bottom face of themicro-plate 20. The bottom faces of the wells 22 of the micro-plate 20are made of optically transparent material such as glass or acrylicresins. Light which has passed through the objective lens 10 enters intothe samples contained in the wells 22, not having hardly attenuatedthrough the immersion medium 31.

In the case of observing/measuring samples using the micro-plate 20 as acontainer, normally, samples are contained in a lot of wells 22. Each ofthe samples is separately measured or observed. Therefore, measurementneeds to be sequentially carried out repeatedly, while operating the XYstage 19 in the XY plane to adjust finely the micro-plate 20. Since themicro-plate 20 is repeatedly moved above the objective lens 10 of whichtip portion is filled with the immersion medium 31, the immersion medium31 formed on the tip portion of the objective lens 10 scatters as themicro-plate 20 moves. If measurement takes a long time, thermal energyof the light is transferred to the immersion medium 31 because the lightpasses through the objective lens 10. This causes evaporation of theimmersion medium 31. Therefore, a sufficient amount of immersion mediumneeds to be filled between the tip portion of the objective lens 10 andthe bottom face of the micro-plate 20.

As described in an example, the immersion medium 31 is not limited towater (immersion water) but oil may be used.

FIG. 3 is a perspective view showing a retention mechanism for animmersion medium, according to the first embodiment. FIG. 4 is alongitudinal cross-sectional view of the retention mechanism for animmersion medium, according to the first embodiment. Referring to FIGS.3 and 4, the retention mechanism for the immersion medium will bedescribed. In this embodiment, as shown in FIG. 4, the immersion mediumretention mechanism is constituted by a hollow bottom plate 41 attachedto be engaged in the periphery of the objective lens 10, and a wall 42put around the bottom plate 41. Further, on the surface of the bottomplate 41, an α portion which is subjected to a water repellent treatmentagainst the immersion medium 31 and a β portion which is subjected towater affinity treatment are provided. This retention mechanism for theimmersion medium forms a liquid pool structure for the immersion medium31. Hereinafter, the retention mechanism for the immersion medium willbe called a “liquid pool plate” 40.

The objective lens 10 is inserted in an opening part at the center ofthe bottom plate 41 of the liquid pool plate 40. Sealing is provided byan O-ring 45, to prevent the immersion medium 31 from leaking from theengagement part between the bottom plate 41 and the objective lens 10.

A hollow liquid feed tube 34 made of vinyl chloride resin is connectedto the wall 42 of the liquid pool plate 40. Through inside the liquidfeed tube 34, the immersion medium 31 is fed onto the objective lens 10.As shown in FIG. 4, the immersion medium 31 covers a lens ring 43 at thetip portion of the objective lens 10, and forms a dome-like mass definedby a contact angle relationship with the a portion.

As the micro-plate 20 moves, the immersion medium 31 moves along thesurface of the α portion and flows down on the surface of the β portion.The immersion medium 31 is stopped by the wall 42 and retained in theliquid pool plate 40. A hollow drain tube 35 made of vinyl chlorideresin is connected to the bottom of the liquid pool plate 40. Throughthis drain tube 35, the immersion medium 31 flowing down inside theliquid pool plate 40 is drained.

The liquid pool plate 40 has a circular outer shape, and materialthereof is aluminum. Material of the wall 42 put around the liquid poolplate 40 is aluminum as well. The wall 42 is 2 mm high and 1 mm thick.Both the liquid feed tube 34 and the drain tube 35 have an outerdiameter of 3 mm and an inner diameter of 2 mm. Material of the liquidpool plate is not limited to aluminum but may be, for example, metalsuch as iron or copper, or alternatively glass, acrylic resin, or thelike. The liquid feed tube 34 and the drain tube are not limited tovinyl chloride resin but may be other resin of polyethylene orpolystyrene, or may alternatively be glass tubes or pipes made of metalsuch as iron.

A basic concept of the retention mechanism for an immersion mediumaccording to the present invention will be described next. FIGS. 5A and5B are views schematically showing a contact angle between a substrateand an immersion medium.

When the affinity is high, wettability is high and the contact angle issmall. When the affinity is low, the wettability is low and the contactangle is large. The present invention utilizes the affinity betweenmaterial retaining the immersion medium and the immersion medium. Bychanging this material, the affinity is controlled so that the immersionmedium can be suitably retained and drained.

Hereinafter, a detailed description will be made of retention anddrainage of the immersion medium along with a specific structurethereof.

A first member (α portion) having a low affinity with the immersionmedium 31 is positioned so as to surround the tip portion of theobjective lens 10. More specifically, if the contact angle between theimmersion medium 31 and the first member is not smaller than apredetermined angle, the immersion medium can be repelled effectively.As a result of this, the immersion medium 31 can be kept in a dome-likeshape by surface tension. Even if a large amount of immersion medium 31is supplied, the immersion medium 31 can be kept in a dome-like shape bysurface tension against gravity. Then, in this state, the immersionmedium 31 can be maintained stably even if observation or measurement iscarried out for a long time with the immersion medium 31 made contactthe bottom face of a sample container such as a micro-plate 20.Therefore, stable measurement data can be obtained.

If water is used as an immersion medium, material which repels theimmersion medium 31, i.e., material having a low affinity is, forexample, resin such as nylon, polyvinyl chloride, polyvinylidenechloride, polyethylene terephthalate, or polymethyl methacrylate.Preferably, the material is polyethylene terephthalate (contact angle:75°) or polymethyl methacrylate (contact angle: 80°). More preferably,the material has a contact angle of 90° or more, and is polyethylene,polystyrene, polycarbonate, polypropylene, polyacetal, various resinsuch as ABS resin, various resin containing fluorine such aspolytetrafluoro ethylene, polytrifluoro ethylene, or polychlorotrifluoroethylene, or ZnO (zinc oxide) or the like. By use of these materials,the member for retaining the immersion medium near the tip portion ofthe objective lens can be constituted.

These materials, alkylsilane, or fluoroalkylsilane are coated to asubstrate directly or indirectly, to retain the immersion medium nearthe tip portion of the objective lens. Further, the member for retainingthe immersion medium near the tip portion of the objective lens can beconstituted by composite plating by which plating is effected on asubstrate while suspending particles of various resin containingfluorine such as polytetrafluoro ethylene, polytrifluoro ethylene, orpolychlorotrifluoro ethylene in an electrolyte.

In order to lower the affinity and expand the contact angle in the caseof using water as the immersion medium, a hydrophobic group such asalkylsilane or fluoro alkylsilane should preferably be formed on thesurface of substrate.

This hydrophobic group may be -R, -RX, —SiR3, —SiRmXn, —OR, —CS,—COOCH3, —CH2OCH3, or the like. In this case, R is an alkyl group and Xis halogen.

Further, in the sample container and a peripheral portion where a liquidoverflowing due to contact of the immersion medium 31 is collected, thesecond member (β portion) is formed to have an affinity with liquid.Therefore, the immersion medium 31 tends to flow easily so that animproved drainage characteristic can be obtained. Specifically, if theaffinity is improved between the second member forming the outercircumference of the first member and the immersion medium 31, thedrainage characteristic can be improved. In particular, the drainagecharacteristic can be improved more if the contact angle is smaller thana predetermined angle.

In order to reduce the contact angle in the case of using water as theimmersion medium, a hydrophilic group should preferably be formed on thesurface of substrate, e.g., water glass or hydrolysate of siliconalkoxide, with or without water-soluble resin or colloidal silica addedthereto, should preferably be formed on the surface of substrate in theperipheral portion where overflowing liquid is collected.

This hydrophobic group is —SO₃H, —SO₃M, —OSO₃H, —OSO₃M, —COOM, —NR₃HX,—COOH, —NH₂, —CN, —OH, —NHCONH₂, or —(OCH₂CH₂)_(n)—. In this case, R isan alkyl group, M is alkaline metal or —NH₄, and X is halogen.

Alternatively, the surface having an affinity with water may be formedby photocatalysis by coating TiO₂ (titanium dioxide).

Various methods are applicable as a surface treatment method capable ofcontrolling the affinity with these immersion media. Various plating,painting, thermal spraying, fusion bonding, coating, dip coating, spincoating, spray coating, vapor deposition, sputtering, or the like may beapplied.

Based on this fundamental technical idea, the retention mechanism for animmersion medium is constituted as follows, according to the firstembodiment.

FIG. 6 is a cross-sectional view of a liquid pool plate. A bottom plate41 and a lens ring 43 are formed of aluminum. Polytetrafluoro ethylenewas baked to be painted on an α portion and the surface of the lens ring43. The contact angle of this polytetrafluoro ethylene to water as theimmersion medium 31 was about 110°. The lens ring 43 has an inner radiusr of 18 mm, an outer diameter R of 30 mm, and an inclination angle θ of15°.

This apparatus was supplied with water as the immersion medium 31. Theobservation position was moved, and the focus adjustment was carriedout. The contact angle of water as the immersion medium 31 to the αportion was large, so that the immersion medium stably maintained adome-like shape effectively between the tip portion of the objectivelens 10 and the bottom face of the micro-plate 20 for a long time.

The present invention is arranged such that, even if a large amount ofimmersion medium 31 is supplied, filled between the tip portion of theobjective lens 10 and the bottom face of the micro-plate 20, theimmersion medium 31 does not easily run down along the main body of theimmersion objective lens 10 due to gravity but maintains stably theimmersion medium 31 at the tip portion of the immersion objective lens10 for a long time.

To achieve this object, the α portion or an area retaining the immersionmedium 31, which is constituted by the lens ring 43, has an innerdiameter r of 2 to 20 mm, an outer diameter R of 10 to 40 mm, and aninclination angle θ ranging from 1 to 30 degrees, as shown in FIG. 6.

[Modification of the First Embodiment]

Next, a modification of the first embodiment will be described. Thismodification has a different retention mechanism for an immersionmedium. FIG. 7 is an enlarged view showing the tip portion of anobjective lens and a part of a micro-plate.

As shown in FIG. 7, in this modification, the inner wall of a liquidpool plate 40 has a conical shape about the objective lens 10 as thecenter thereof. Meanwhile, the outer wall of the liquid pool plate 40has a cylindrical shape. Even if an immersion medium fed from anautomatic liquid feed/drain mechanism 24 overflows from the lens part atthe center of the objective lens 10, the immersion medium can besmoothly guided into a drain tube 35 because the inner wall of theliquid pool plate 40 has a conical shape.

[Modification 2 of the First Embodiment]

FIG. 8 is a view from upside, showing a lens ring portion retaining animmersion medium. In this modification, the lens ring portion has aseparate structure consisting of an α member and a β member, as shown inFIG. 8. This enables suitable maintenance and drainage of the medium.

In further another modification, the retention mechanism for animmersion medium is constituted by a lens ring portion, a hollow bottomplate 41 attached to be engaged around the objective lens 10, and a wall42 put along the periphery of the bottom plate 41, as shown in FIG. 9.The lens ring portion means the whole inclined face from the lens at thetip portion of the objective lens to an outer frame of the lens. Thelens ring portion is made of aluminum and has a recess in which an amember is engaged. The whole lens ring portion is subjected to analumite treatment. Polypropylene (at a contact angle of about 90°) asmaterial having a low affinity with water was molded into a donut-likeshape, which was fitted in the recess. Alumite has a higher affinitywith water than polypropylene. The immersion medium was maintainedstably on the lens, and excess of the immersion medium was drainedsmoothly.

[Second Embodiment]

The retention mechanism for an immersion medium according to the secondembodiment of the present invention has the same shape as that of thefirst embodiment. However, the surface treatment method for treating theα portion, β portion, and lens ring 43 is different from that of thefirst embodiment. Therefore, the same portions as those of the firstembodiment will be denoted at the same reference symbols, and detaileddescriptions thereof will be omitted herefrom.

According to the second embodiment, the α portion and the lens ring 43were subjected to spray coating of a methanol solution containing 2%CF₃CF₂CH₂Si(OCH₃)₃ as a kind of fluoroalkylalkoxysilane while the βportion was masked to be not coated. Thereafter, the resultant was driedat a room temperature.

Next, the mask was removed from the β portion. With the α portionmasked, a small amount of nitric acid was added to a methanol solutionof trimethoxysilane, and hydrolysis occurred for an hour. Thereafter,the hydrolysis solution was spray coated on the β portion. The resultantwas dried for an hour at 120° C. Before coating, the α portion beforecoating has an average surface roughness of 2 nm. Contact angles of theα portion and the β portion to water according to the present embodimentwere measured to be about 60° and 20°, respectively.

This apparatus was supplied with water as an immersion medium 31. Theobservation position was moved, and the focus was adjusted. A dome ofwater as the immersion medium 31 was effectively formed and maintained.Water which ran down from the dome was effectively drained. Thus, it hasbeen confirmed that the dome of water is effectively formed and retainedstably and constantly if the contact angle of the immersion medium tomaterial having a low affinity is 60°, and that water running down fromthe dome is effectively drained.

[Third Embodiment]

The retention mechanism for an immersion medium according to the thirdembodiment of the present invention has the same shape as that of thefirst embodiment. However, the surface treatment method for treating theα portion, β portion, and lens ring 43 is different from that of thefirst embodiment. Therefore, the same portions as those of the firstembodiment will be denoted at the same reference symbols, and detaileddescriptions thereof will be omitted herefrom.

According to the third embodiment, the α portion and the lens ring 43were subjected to spray coating of a methanol solution containing 2%CF₃CF₂CH₂Si(OCH₃)₃ as a kind of fluoroalkylalkoxysilane while the βportion was masked to be not coated. Thereafter, the resultant was driedat a room temperature.

Next, the mask was removed from the β portion. With the α portionmasked, a small amount of nitric acid was added to a methanol solutionof trimethoxysilane, and hydrolysis occurred for an hour. Thereafter,the hydrolysis solution was spray coated on the β portion. The resultantwas dried for an hour at 120° C. Before coating, the α portion beforecoating has average surface roughness of 14 nm. Contact angles of the αportion and the β portion to water according to the present embodimentwere measured to be about 100° and 20°, respectively.

This apparatus was supplied with water as an immersion medium 31. Theobservation position was moved, and the focus was adjusted. A dome ofwater as the immersion medium 31 was effectively formed and maintained.Water which ran down from the dome was effectively drained.

[Fourth Embodiment]

FIG. 10 is a view from upside, showing a lens ring portion retaining animmersion medium. In the fourth embodiment, as shown in FIG. 10, thesurface of the lens ring portion is constituted by three kinds ofmaterials, i.e., an α portion, β portion, and γ portion. The α portionis the closest portion to the center axis of the objective lens and usesmaterial having an affinity with water. The β portion is formed like aring around the outer circumference of the α portion, and uses materialhaving a water repellent characteristic. The γ portion is positioned atthe outermost part of the objective lens, and uses material having anaffinity with water.

Alternatively, the tip portion of the retention mechanism may use onekind of member, e.g., aluminum. The surface part thereof may be appliedor coated with different members, i.e., α portion, β portion, and γportion. In this case, the α portion is applied or coated with materialhaving an affinity with water. The β portion is applied or coated withmaterial having a water repellent characteristic. For the γ portion, thetip portion of the retention mechanism is applied or coated withmaterial having an affinity with water.

Further, the α portion, β portion, and γ portion contact each othertightly, constituting the tip portion of the retention mechanism for animmersion medium. The α portion, β portion, and γ portion each has adownward inclination from the center axis of the objective lens towardthe periphery. By this structure, excessive immersion water isimpregnated into the liquid pool plate 40.

Specifically, a SiO₂—TiO₂ film should preferably be formed as a filmhaving an affinity with water, at the α portion and γ portion. This filmis capable of maintaining the surface given an affinity with water, dueto photocatalysis. Even a one-component film of titania has a surfacehaving an affinity with water due to photocatalysis. This is because thephotocatalysis may cease, in some cases, to degrade the affinity withwater while illumination light is not irradiated. In contrast, aSiO₂—TiO₂ two-component film is capable of maintaining the surfaces ofthe α portion and γ portion given a proper affinity with water, incooperation with the affinity of the Si—OH group to water, while lightis not irradiated.

Since light is often shut out during measurement, light shouldpreferably be irradiated to recover the affinity with water or a lightsource such as a lamp should preferably be provided to recover theaffinity with water, after measurement.

The SiO₂—TiO₂ two-component film can be prepared, for example, by asol-gel method. For example, this film is prepared as follows. Siliconalkoxide is diluted with alcohol, stirred, and subjected to partialhydrolysis. The resultant thereof is thereafter added with titaniumalkoxide diluted with alcohol, and is further added with a smallquantity of water for final hydrolysis. Further, the resultant isstirred to adjust the sol. The sol thus obtained is applied, with the βportion masked, and is then subjected to a heat treatment. A SiO₂—TiO₂two-component film is formed at a desired portion.

Although various methods are available as the stirring method at thetime of adjusting the sol, use of a planetary stirring/defoaming deviceis preferred. If a planetary stirring/defoaming device is used, reactionproceeds so uniformly that the partial hydrolysis can be controlledeasily, in particular. Film formation can be achieved with uniformcharacteristics. Besides, the sol can be adjusted within a very shorttime period.

Next, the mask is removed from the β portion. The α portion and γportion are masked. The β portion is spray-coated with a methanolsolution containing 2% CF₃CF₂CH₂Si(OCH₃)₃ as a kind offluoroalkylalkoxysilane. The resultant is dried at a room temperature.

In another structure, as shown in FIG. 11, the α portion and β portionare provided in tight contact with each other, forming the tip portionof the retention mechanism for an immersion medium. At the part of theliquid pool plate 40, a γ portion may be applied or coated.Alternatively, the liquid pool plate 40 is subjected to a surfacetreatment to produce the γ portion.

By dividing the lens ring portion into separate bands, e.g., an αportion having an affinity with water, a β portion having a waterrepellent characteristic, and a γ portion having an affinity with water,orderly from the center. As a result of this, the liquid quantity of theimmersion medium retained on the lens at the tip portion of theobjective lens, and the lens ring portion in the periphery thereof isgreater than those of the two band structures, i.e., the α portion andthe β portion. Therefore, a much greater quantity of immersion mediumcan be maintained at the tip portion of the objective lens,advantageously when carrying out a microscopic observation for a longtime. In addition, the immersion medium needs to be charged lessfrequently, so that experimenters can save labor.

The retention mechanism for an immersion medium is constituted by ahollow bottom plate attached in engagement with the periphery of theobjective lens 10, and a wall put around along the periphery of thebottom plate. Alternatively, the mechanism is constituted by a lens ringportion, a hollow bottom plate attached in engagement with the peripheryof the objective lens 10, and a wall put around along the periphery ofthe bottom plate.

The immersion medium 31 is not limited to water immersion. The object ofthe present invention can be achieved by the same structures in case ofan oil immersion method. At this time, attention should be paid to thataffinities of respective members with the immersion medium 31 in thecase of using oil are opposite to those in the case of using water. Forexample, in the description made above, material which has a highaffinity in the case of using water as the immersion medium 31 can beused as material having a low affinity in the oil immersion method.Inversely, material which has a low affinity when water is used as theimmersion medium 31 can be used as material having a high affinity inthe oil immersion method.

The present invention is not limited directly to the above embodimentsbut may be practiced with constitutional elements thereof modifiedwithout deviating from the subject matter of the invention in practicalphases. Further, various inventions may be formed by appropriatelycombining plural constitutional elements disclosed in any of the aboveembodiments. For example, several constitutional elements may be removedfrom all constitutional elements suggested in any of the embodiments.Furthermore, constitutional elements may be combined between differentembodiments.

What is claimed is:
 1. A retention mechanism for an immersion medium,for use in a device which observes/measures a sample by use of animmersion objective lens, the immersion objective lens being formed of atip portion, a lens ring portion and a lens outer frame, the lens ringportion being formed of an entire surface inclined from the lens of thetip portion to the lens outer frame, the retention mechanism comprising:a bottom plate which has an opening portion to be engaged around theimmersion objective lens; a wall plate arranged along a periphery of thebottom plate; an immersion medium supply unit which supplies theimmersion medium to the tip portion of the immersion objective lens; anda drip draining unit of the immersion medium provided on the bottomplate in vicinity of the wall plate, which drains a drip of theimmersion medium from the tip portion of the immersion objective lens,the bottom plate comprising a first ring-member formed of a firstmaterial for retaining the immersion medium and arranged to surround theopening portion, the bottom plate being inclined to make a side of theopening portion higher than a side of the wall plate, an affinity of thefirst ring-member with the immersion medium being lower than an affinityof an inner member of the first ring-member with the immersion medium.2. The retention mechanism for an immersion medium, according to claim1, further comprising a second ring-member formed of a second materialfor draining the immersion medium, which is arranged outside the firstring-member to surround the first ring-member, wherein the affinity ofthe first ring-member with the immersion medium is lower than anaffinity of the second ring-member with the immersion medium.
 3. Theretention mechanism for an immersion medium, according to claim 2,wherein a contact angle between the first ring-member and the immersionmedium is greater than 60°.
 4. The retention mechanism for an immersionmedium, according to claim 3, wherein the material of at least one ofthe first ring-member retaining the immersion medium and the secondring-member is synthetic resin.
 5. The retention mechanism for animmersion medium, according to claim 1, wherein a contact angle betweenthe first ring-member and the immersion medium is greater than 60°. 6.The retention mechanism for an immersion medium, according to claim 5,wherein the first ring-member retaining the immersion medium is formedof synthetic resin.
 7. The retention mechanism for an immersion medium,according to claim 6, wherein the first ring-member is a regionsurface-processed with a material for retaining the immersion medium. 8.The retention mechanism for an immersion medium, according to claim 1,wherein the first ring-member retaining the immersion medium is formedof synthetic resin.
 9. The retention mechanism for an immersion medium,according to claim 8, wherein the first ring-member is a regionsurface-processed with a material for retaining the immersion medium.10. The retention mechanism for an immersion medium, according to claim1, wherein the first ring-member is a region surface-processed with amaterial for retaining the immersion medium.
 11. A method ofmanufacturing a retention mechanism for an immersion medium, for use ina device which observes/measures a sample by use of an immersionobjective lens, the immersion objective lens being formed of a tipportion, a lens ring portion and a lens outer frame, the lens ringportion being formed of an entire surface inclined from the lens of thetip portion to the lens outer frame, the method comprising: forming anopening portion to be engaged around the immersion objective lens, on abottom plate; arranging a wall plate along a periphery of the bottomplate; providing an immersion medium supply unit which supplies theimmersion medium to the tip portion of the immersion objective lens:providing a drip draining unit of the immersion medium which drains adrip of the immersion medium from the tip portion of the immersionobjective lens, on the bottom plate in vicinity of the wall plate; andarranging a first ring-member formed of a first material for retainingthe immersion medium, on the bottom plate, so as to surround the openingportion, the bottom plate being inclined to make a side of the openingportion higher than a side of the wall plate, an affinity of the firstring-member with the immersion medium being lower than an affinity of aninner member of the first ring-member with the immersion medium.
 12. Themethod according to claim 11, further comprising arranging a secondring-member formed of a second material for draining the immersionmedium, which is arranged outside the first ring-member to surround thefirst ring-member, wherein the affinity of the first ring-member withthe immersion medium is lower than an affinity of the second ring-memberwith the immersion medium.
 13. The method according to claim 12, whereina contact angle between the first ring-member and the immersion mediumis greater than 60°.
 14. The method according to claim 13, wherein thematerial of at least one of the first ring-member retaining theimmersion medium and the second ring-member is synthetic resin.
 15. Themethod according to claim 11, wherein a contact angle between the firstring-member and the immersion medium is greater than 60°.
 16. The methodaccording to claim 15, wherein the first ring-member retaining theimmersion medium is formed of synthetic resin.
 17. The method accordingto claim 16, wherein the first ring-member is a region surface-processedwith a material for retaining the immersion medium.
 18. The methodaccording to claim 11, wherein the first ring-member retaining theimmersion medium is formed of synthetic resin.
 19. The method accordingto claim 18, wherein the first ring-member is a region surface-processedwith a material for retaining the immersion medium.
 20. The methodaccording to claim 11, wherein the first ring-member is a regionsurface-processed with a material for retaining the immersion medium.